Long Noncoding RNA MIR100HG Knockdown Attenuates Hepatocellular Carcinoma Progression by Regulating MicroRNA-146b-5p/Chromobox 6

Abstract

Purpose: Hepatocellular carcinoma (HCC) accounts for approximately ninety percent of primary liver cancer. This study attempted to investigate the effects of the long noncoding RNA MIR100HG (MIR100HG) in HCC and the underlying molecular mechanism.

Materials and methods: qRT-PCR was implemented to analyze the expression of MIR100HG, microRNA-146b-5p (miR-146b-5p), and Chromobox 6 (CBX6). The correlation between MIR100HG and clinicopathological features of HCC patients was assessed. Additionally, the effects of MIR100HG knockdown on HCC cell viability, migration, and invasion were explored. The interactions among MIR100HG, miR-146b-5p, and CBX6 were confirmed. Furthermore, rescue experiments were conducted to investigate whether MIR100HG knockdown modulates HCC cell behaviors through modulating the miR-146b-5p/CBX6 axis.

Results: The expression of MIR100HG and CBX6 was enhanced, while miR-146b-5p was inhibited in HCC cells. High MIR100HG expression was positively associated with the TNM tumor stage and Edmondson-Steiner grading in HCC patients. MIR100HG knockdown considerably reduced the HCC cell viability, migration, and invasion. In addition, MIR100HG directly targeted miR-146b-5p, and miR-146b-5p directly targeted CBX6 in HCC cells. Moreover, miR-146b-5p suppression or CBX6 elevation evidently rescued the suppressed viability, migration, and invasion of HCC cells caused by MIR100HG knockdown.

Conclusions: Knockdown of MIR100HG inhibited the viability, migration, and invasion of HCC cells by targeting the miR-146b-5p/CBX6 axis, offering a potential therapeutic target for HCC therapy.

Figure 1

The expression of MIR100HG was enhanced in hepatocellular carcinoma (HCC) tissues and cells. (a) The expression of MIR100HG in HCC tumor tissues and adjacent tissues was detected by qRT-PCR. ^∗∗∗P < 0.001 vs. normal. (b) Relative expression of MIR100HG in HCC patients at the TNM I/II and TNM III/IV. ^∗∗P < 0.01 vs. I/II. (c) qRT-PCR was performed to measure the expression of MIR100HG in LO2, Hep3B, HepG2, SK-HEP1, and Huh7 cells. ^∗∗∗P < 0.001 vs. LO2.

Figure 2

MIR100HG knockdown attenuated the tumorigenesis of hepatocellular carcinoma (HCC) cells. (a) The transfection efficiency of si-NC, si-MIR100HG-1, and si-MIR100HG-2 in Hep3B and SK-HEP1 cells was evaluated by qRT-PCR. ^∗∗P < 0.01, ^∗∗∗P < 0.001 vs. si-NC. (b) The viability of Hep3B and SK-HEP1 cells was measured by the MTT assay. ^∗∗∗P < 0.001 vs. si-NC. (c, d) The migration and invasion of Hep3B and SK-HEP1 cells were determined by the wound healing assay and invasion assay. ^∗∗P < 0.01, ^∗∗∗P < 0.001 vs. si-NC.

Figure 3

miR-146b-5p was directly targeted by MIR100HG. (a) starBase displayed the predicted binding site between MIR100HG and miR-146b-5p. (b) Relative luciferase activity in Hep3B and SK-HEP1 cells was measured by the dual-luciferase reporter assay. ^∗∗P < 0.01 vs. mimic NC. (c) The expression of miR-146b-5p was upregulated by the transfection of si-MIR100HG-1 in Hep3B and SK-HEP1 cells. ^∗∗∗P < 0.001 vs. si-NC. (d) qRT-PCR was performed to determine the expression of miR-146b-5p in hepatocellular carcinoma (HCC) tumor tissues and adjacent tissues. ^∗∗P < 0.01 vs. normal. (e) The expression of MIR100HG was negatively correlated with miR-146b-5p in HCC tumor tissues. (f) The expression of miR-146b-5p in LO2, Hep3B, HepG2, SK-HEP1, and Huh7 cells was detected by qRT-PCR. ^∗∗∗P < 0.001 vs. LO2.

Figure 4

miR-146b-5p restrained the tumorigenesis of hepatocellular carcinoma (HCC) cells. (a) The transfection efficiency of mimic NC, miR-146b-5p mimics, inhibitor NC, and miR-146b-5p inhibitor was measured by qRT-PCR in Hep3B and SK-HEP1 cells. ^∗∗∗P < 0.001 vs. mimic NC, ^###P < 0.001 vs. inhibitor NC. (b) MTT assay was performed after transfection with mimic NC or miR-146b-5p mimics in Hep3B and SK-HEP1 cells. ^∗∗∗P < 0.001 vs. mimic NC. (c, d) The effects of miR-146b-5p elevation on the migration and invasion of Hep3B and SK-HEP1 cells were evaluated. ^∗∗P < 0.01, ^∗∗∗P < 0.001 vs. mimic NC.

Figure 5

miR-146b-5p directly targeted CBX6. (a) TargetScan exhibited the predicted binding site between CBX6 and miR-146b-5p. (b) Dual-luciferase reporter assay was performed to measure the relative luciferase activity in Hep3B and SK-HEP1 cells. ^∗∗∗P < 0.001 vs. mimic NC. (c) The protein expression of CBX6 in Hep3B and SK-HEP1 cells was measured by western blot. ^∗∗∗P < 0.001 vs. mimic NC. (d) qRT-PCR was used to detect the expression of CBX6 in hepatocellular carcinoma (HCC) tumor tissues and adjacent tissues. ^∗∗P < 0.01 vs. normal. (e) The expression of CBX6 was negatively correlated with miR-146b-5p in HCC tissues. (f) The expression of CBX6 was positively correlated with MIR100HG in HCC tissues. (g) qRT-PCR was performed to evaluate the expression of CBX6 in LO2, Hep3B, HepG2, SK-HEP1, and Huh7 cells. ^∗∗P < 0.01 vs. LO2.

Figure 6

MIR100HG deficiency suppressed the tumorigenesis of hepatocellular carcinoma (HCC) cells by targeting the miR-146b-5p/CBX6 axis. (a) The transfection efficiency of pcDNA-NC and pcDNA-CBX6 was measured by qRT-PCR in Hep3B cells. ^∗∗∗P < 0.001 vs. pcDNA-NC. (b) Western blot was performed to measure the protein expression of CBX6 in Hep3B cells. ^∗∗∗P < 0.001 vs. si-NC, ^###P < 0.001 vs. si-MIR100HG-1. (c–e) Inhibition of miR-146b-5p or overexpression of CBX6 reversed the inhibitory effects of MIR100HG knockdown on viability, migration, and invasion of Hep3B cells. ^∗∗P < 0.01, ^∗∗∗P < 0.001 vs. si-NC; ^##P < 0.01 vs. si-MIR100HG-1.